L-threonine and L-tryptophan producing bacteria strain and method of making same

ABSTRACT

The present invention relates to a microorganism able to produce L-threonine or L-tryptophan, and to a method for producing L-threonine or L-tryptophan by using same. More specifically, the present invention relates to: recombinant  Escherichia coli  which is more efficient in producing L-threonine or L-tryptophan by increasing the ability to produce ATP which is used as the most plentiful energy source in cells when producing L-threonine or L-tryptophan; and a method for producing L-threonine or L-tryptophan by using same.

This application is a National Stage Entry of International ApplicationNo. PCT/KR2013/000072, filed on Jan. 7, 2013, and claims the benefit ofKorean Application No. 10-2012-0001819, filed on Jan. 6, 2012, which arehereby incorporated by reference in their entirety for all purposes asif fully set forth herein.

TECHNICAL FIELD

The present invention relates to a microorganism able to produceL-threonine or L-tryptophan and to a method of producing L-threonine orL-tryptophan using the same.

BACKGROUND ART

Incorporated by reference herein in its entirety is the Sequence Listingentitled “sequence Listing.txt”, created Jul. 2, 2014, size of 11kilobytes.

It is known that microorganisms which produce useful products throughfermentation require very large amounts of energy such as ATP when thebiosynthetic pathway is enhanced.

As is known in the art, it is very important that the intracellularbalance between nicotinamide adenine dinucleotide (NAD(H)) that isproduced by catabolic reactions and nicotinamide adenine dinucleotidephosphate NADP(H) that is used in anabolic reactions in microbialmetabolic processes. NAD(H) is an intermediate in the catabolicreactions that generate ATP through the oxidation of food and functionsas an energy source. And NADP(H) play roles in providing a reducingpower in the in vivo metabolic process, that is providing thehigh-energy electrons needed to synthesize molecules by reacting withenzyme which generally catalyze an anabolic reactions. The balancetherebetween is regulated either by the phosphorylation of NAD as shownin the following equation 1) or by the dephosphorylation of NADP asshown in the following equation 2).NAD⁺+ATP

NADP⁺+ADP  Equation 1)NADP⁺

NAD⁺+phosphate  Equation 2)

Thus, in order to effectively produce reducing power such as NADPH, aphosphate source such as ATP should be increased together.

ATP (Adenosine-5′-triphosphate) has a high energy phosphate bond, andgenerates energy when it is hydrolyzed to ADP and phosphate. ATP isproduced mainly by chemiosmotic phosphorylation via the electrontransport system in microorganisms or by substrate-levelphosphorylation. The produced ATP is degraded to supply the energyrequired for cells and is reused by regenerating via glycolysis pathwayor oxidative phosphorylation.

Based on this fact, studies have been conducted to apply bacteria's ATPenergy regenerating process to the mass production of useful products inorder to facilitate energy supply (Biosci Biotechnol Biochem., (1997)61: 840-845). In studies on the regeneration of ATP in E. coli, it wasfound that the level of ATP in a microorganism is about 150% higher thanthat in the parent strain when a few genes, including ysaA (NCBI GeneID: 948085), ydaS (NCBI Gene ID: 945923) and ybiX (NCBI Gene ID: 947502)genes was deficient, respectively, and this finding was applied to theproduction of glutathione (FEMS Microbiol Lett., (2009) 297:217-224).However, there was no direct report directly explains the increase inproduction of amino acids caused by attenuation in the activities ofproteins that are encoded by the genes.

DISCLOSURE Technical Problem

The present inventors have found that increasing the intracellular levelof ATP, which is used as the most plentiful energy source in cellsproducing L-amino acid, is effective to increase the production ofL-threonine or L-tryptophan, thereby completing the present invention.

An object of the present invention is to provide a recombinant E. colistrain which has an increased L-threonine or L-tryptophan productivityby increasing the productivity of ATP.

Another object of the present invention is to provide a method ofproducing L-threonine or L-tryptophan using the recombinant E. colistrain.

Technical Solution

In order to accomplish the above objects, an embodiment of the presentinvention provides an L-threonine or L-tryptophan producing recombinantE. coli strain, wherein the strain is modified to attenuate (weaken)activity of at least one protein selected from the group consisting of aprotein YsaA having an amino acid sequence represented by SEQ ID NO: 2,a protein YdaS having an amino acid sequence represented by SEQ ID NO:4, and a protein YbiX having an amino acid sequence represented by SEQID NO: 6.

An embodiment of The present invention also provides a method forproducing L-threonine or L-tryptophan, which comprises culturing therecombinant E. coli strain.

Advantageous Effects

The present invention provides a recombinant microorganism whoseL-threonine or L-tryptophan productivity is improved by increasing theintracellular ATP level in a microorganism having L-threonine orL-tryptophan productivity. According to the present invention, itprovides a method to enhance production of L-threonine or L-tryptophanby recovering the balance of energy metabolism to increase the cellularactivity and reduce the culture time.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the relative ATP level (%) of an L-threonine-producingstrain relative to that of its parent strain.

FIG. 2 shows the relative ATP level (%) of a L-tryptophan-producingstrain relative to that of its parent strain.

BEST MODE

Hereinafter, the present invention will be described in detail.

An embodiment of the present invention provides an L-threonine orL-tryptophan producing recombinant E. coli strain, wherein the strain ismodified to attenuate activity of at least one selected from the groupconsisting of a protein YsaA having an amino acid sequence representedby SEQ ID NO: 2, a protein YdaS having an amino acid sequencerepresented by SEQ ID NO: 4, and a protein YbiX having an amino acidsequence represented by SEQ ID NO: 6.

An L-threonine or L-tryptophan producing microorganism that may be usedin the present invention may be any microorganism capable of producingL-threonine or L-tryptophan, such as Escherichia sp. Bacterium, E. coli,Coryneform bacterium, Serratia sp. bacterium, Providencia sp. bacterium,or the like. Specifically, a microorganism belonging to the genusEscherichia may be used.

In a specific embodiment of the present invention, recombinant E. colistrain CJ600 (KCCM 10812P) (Korean Patent Registration No. 10-0792095)having L-tryptophan productivity is used, which is obtained bygenetically engineering an recombinant E. coli strain (KFCC 10066)having L-phenylalanine productivity so as to desensitize the tryptophanauxotrophy, block L-phenylalanine biosynthesis and enhance tryptophanbiosynthesis-related genes.

In another specific embodiment of the present invention, recombinant E.coli strain FTR2533 (KCCM-10541) (Korean Patent Registration No.10-0576342) having L-threonine productivity is used, which is obtainedby genetically engineering an E. coli mutant strain (KFCC 10718) havingL-threonine productivity so as to inactivate the wild-type galR gene.

YsaA, a protein having an amino acid sequence represented by SEQ ID NO:2, is predicted a hydrogenase of 4Fe-4S ferredoxin-type component, butits exact function has not yet been found.

YdaS, a protein having an amino acid sequence represented by SEQ ID NO:4, is predicted a DNA binding transcription regulator, but its exactfunction has not yet been found.

YbiX, a protein having an amino acid sequence represented by SEQ ID NO:6, is one of the Fe(II)-dependent oxygenase superfamily, which functionsas an oxidoreductase that oxidizes its substrate using oxygen.

The polypeptides YsaA, YdaS and YbiX of the present invention have theamino acid sequences represented by SEQ ID NOS: 2, 4 and 6,respectively, but are not limited thereto, because the amino acidsequences of the proteins may depending on the species or strains ofmicroorganisms.

In other words, the proteins of the present invention may be mutants orartificial variants encoding a protein that has an amino acid sequenceincluding a substitution, deletion, insertion, addition or inversion ofone or several amino acids in one or more positions of the amino acidsequence represented by SEQ ID NO: 2, 4 or 6, as long as the mutants orartificial variants can be helpful in increasing the production of aminoacid by attenuating the activities described in the present invention.Herein, the number of “several” amino acids differs depending on theposition or type of amino acid residues in the three-dimensionalstructure of the protein, but is particularly 2-20, specifically 2-10,and more specifically 2-5. In addition, this substitution, deletion,insertion, addition or inversion of amino acids also include thosealterations caused by a naturally occurring mutation or artificialvariation based on the difference in individuals or species ofmicroorganisms having the activity of the polypeptides.

As used herein, the term “attenuation” means that the activity of aprotein is weakened either by deleting part or all of the gene encodingthe protein, or by modifying an expression regulatory sequence to reducethe expression of the gene, or by modifying the chromosomal genesequence to weaken the activity of the protein, or by combinationsthereof.

In the present invention, attenuation of the activity may be achieved bya method selected from the group consisting of: 1) deleting part or allof a polynucleotide encoding the protein; 2) modifying an expressionregulatory sequence to reduce the expression of the polynucleotide; 3)modifying the chromosomal polynucleotide sequence to weaken the activityof the protein; and 4) combinations thereof.

The method for deletion of part or all of the polynucleotide encodingthe protein may be performed by replacing a polynucleotide which encodesan endogenous target protein in the chromosome, with either apolynucleotide that a part of nucleic acid sequence is deleted or amarker gene through chromosome insertion vector.

Herein, the term “a part of” nucleic acid sequence differ depending onthe kind of gene, but is regardless of the position thereof, and it isspecifically 1-200, more specifically 1-100, and even more specifically1-50.

Also, the method of modifying the expression regulatory sequence toreduce the expression of the polynucleotide may be performed either byinducing a mutation in the expression regulatory sequence by thedeletion, insertion, non-conservative or conservative substitution, orcombinations thereof, of one or more nucleotides to attenuate theactivity of the expression regulatory sequence, or by replacing theexpression regulatory sequence with weaker activity. The expressionregulatory sequence includes a promoter, an operator sequence, asequence encoding a ribosome-binding site, a sequence regulating thetermination of transcription and translation.

In addition, the method of modifying the chromosomal polynucleotidesequence encoding the protein of the present invention may be performedeither by inducing a mutation in the sequence by the deletion,insertion, non-conservative or conservative substitution, orcombinations thereof, of one or more nucleotides to attenuate theactivity of the sequence, or by replacing the sequence with an modifiednucleotide sequence having weaker activity.

The polynucleotide encoding the protein of the present invention can beintroduced into a host cell and may be substituted with a codondifficult to express in the host. In addition, the N-terminus orC-terminus thereof may be extended or deleted, and the start codon maybe modified to regulate the expression level.

Each of the polynucleotides of the present invention may have apolynucleotide sequence encoding a protein having a homology of at least80%, specifically at least 90%, more specifically at least 95%, and evenmore specifically at least 97% to the amino acid sequence of eachrepresented by SEQ ID NOS: 2, 4 and 6, as long as the polynucleotide canattenuate the protein activity of the variant. More specifically, thepolynucleotides have a polynucleotide sequence represented by SEQ IDNOs: 1, 3 and 5, respectively.

As used herein, the term “homology” refers to the identity between twoamino acid sequences. The homology can be determined using well-knownmethods, for example, the computer program BLAST 2.0 which calculatesparameters like as score, identity, and similarity.

Also, the polynucleotide sequences of the present invention may behybridized with the polynucleotide sequences represented by SEQ ID NOS:1, 3 and 5 and probes produced from the above-described nucleotidesequences under stringent conditions, and may be modified sequencesencoding normally functioning proteins.

As used herein, the term “stringent conditions” refers to conditionsthat allow specific hybridization between polynucleotides.Alternatively, the term is related to polypeptides or proteins,including derivatives thereof (Molecular Cloning, A Laboratory Manual,J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratorypress, Cold Spring Harbor, N.Y., 1989; or Current Protocols in MolecularBiology, F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., NewYork).

Specifically, the “stringent conditions” refer to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH 7),0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodiumcitrate at pH 7. After hybridization, the membrane to which the DNA hasbeen transferred is washed in 2×SSC at room temperature and then in0.1-0.5×SSC/0.1×SDS at 68° C.

As used herein, the term “vector” refers to a DNA construct containingthe nucleotide sequence of a target protein-encoding gene operablylinked to a suitable regulatory sequence so as to be able to express thetarget gene in a suitable host cell. The regulatory sequence includes apromoter capable of initiating transcription, any operator forregulating this transcription, a sequence encoding a suitable mRNAribosome binding site, and a sequence for regulating the termination oftranscription and translation. Once transformed into a suitable host,the vector may replicate or function independently of the host genome,or may, in some cases, integrate into the genome itself.

The vector that is used in the present invention is not specificallylimited and may be any vector known in the art, as long as it canreplicate in a host. Examples of the commonly used vectors may includenatural or recombinant plasmids, cosmids, viruses, and bacteriophages.For example, the phage vector or cosmid vectors include pWE15, M13,λBL3, λBL4, λXII, λSHII, λPII, λ10, λ11, Charon4A, and Charon21A, andthe plasmid vectors include pBR, pUC, pBluescriptII, pGEM, pTZ, pCL1920and pET-type plasmids. Vectors that may be used are not particularlylimited, and any known expression vectors may be used. Specifically,pACYC177, pACYC184, pCL1920, pECCG117, pUC19, pBR322, pMW118 or pCC1BACvectors may be used. Most specifically, pACYC177, pCL1920 and pCC1BACvectors may be used.

Further, the vector that is used in the present invention is a vectorcapable of transforming host cells, to insert the polynucleotideencoding the target protein into the chromosome of the host cell.Specific examples of the vector include, but are not limited to, theshuttle vector pECCG112 that can self-replicate in both directions in E.coli and Coryne-type bacteria (Kap-Soo, Noh, Kor. Jour. Microbiol. July1991, p 149-154).

Also, the polynucleotide encoding the endogenous target protein in thechromosome can be replaced with a new polynucleotide by a vector forinsertion into the bacterial chromosome. Insertion of the polynucleotideinto the chromosome can be performed by any method known in the art, forexample, homologous recombination. Because the vector of the presentinvention can be inserted into the chromosome by homologousrecombination, it may further comprise a selection marker for confirmingits insertion into the chromosome. The selection marker is used toselect a cell transformed with the vector, that is, confirm theinsertion of the target polynucleotide. The selection marker that isused in the present invention may be selected from markers that provideselectable phenotypes, such as drug resistance, auxotrophy, resistanceto cytotoxic agents, or surface protein expression. Only cellsexpressing the selection marker are able to survive or to show differentphenotypes under the environment treated with the selective agent, andthus the transformed cells can be selected.

As used herein, the term “transformation” means introducing a vectorcomprising the polynucleotide encoding the target protein into a hostcell so as to be able to express the protein encoded by thepolynucleotide in the host cell. The transformed polynucleotides includeall the genes inserted in the chromosome of the host cell or locatedoutside the chromosome, as long as they can be expressed in the hostcell. In addition, the polynucleotides include DNA and RNA, which encodethe target protein. As long as the polynucleotide can be introduced inthe host cell and expressed therein, the gene may be introduced in anyform.

For example, the polynucleotide can be introduced into the host cell inthe form of an expression cassette which is a polynucleotide constructincluding all elements for expressing the gene. The expression cassetteincludes a promoter which is operably linked to the gene, atranscription termination signal, a ribosome binding site, and atranslation termination signal. The expression cassette may be in theform of an expression vector capable of self-replicating. Thepolynucleotide may also be introduced into the host cell by itself, andbe operably linked to the sequence necessary for expression in the hostcell.

Specifically, attenuation of the activity of the protein that is encodedby the ysaA, ydaS or ybiX gene may be achieved by deletion of the gene.Specifically, a mutation in the gene can be induced using chemicals orlight such as UV light, thereby obtaining a variant having the deletedgene. Alternatively, a variant lacking the activity of the protein canbe obtained by substituting the chromosomal gene to the nucleotidelacking the activity by a gene recombination technique, by a method ofgene replacement through homologous recombination.

Also, an embodiment of the present invention also provides a method forproducing L-threonine or L-tryptophan, the method comprising culturingan L-threonine or L-tryptophan producing recombinant E. coli strain,wherein the strain is modified to attenuate activity of at least oneselected from the group consisting of a protein YsaA having an aminoacid sequence represented by SEQ ID NO: 2, a protein YdaS having anamino acid sequence represented by SEQ ID NO: 4, and a protein YbiXhaving an amino acid sequence represented by SEQ ID NO: 6.

The culture process of the present invention can be performed insuitable media and culture conditions known in the art. This cultureprocess can be easily modified by any person skilled in the artdepending on the type of strain selected. Examples of the cultureprocess include, but are not limited to, batch culture, continuousculture, and fed-batch culture.

The media and culture conditions that are used in culture of themicroorganism of the present invention may be those as long as that areused in culture of microorganisms belonging to the genus Escherichia,but these should properly satisfy the requirements of the microorganismof the present invention.

In a specific embodiment of the present invention, the microorganism maybe cultured in a conventional medium containing suitable carbon sources,nitrogen sources, amino acids, vitamins and the like under aerobicconditions while adjusting temperature, pH and the like.

Carbon sources that may be used in the present invention includecarbohydrates such as glucose, fructose, sucrose, maltose, mannitol,sorbitol; alcohols such as sugar alcohol, glycerol, pyruvic acid, lacticacid and citric acid; and amino acids such as organic acid, glutamicacid, methionine and lysine. In addition, natural organic nutrientsources such as starch hydrolysates, molasses, blackstrap molasses, ricebran, cassava, bagasse and corn steep liquor may be used. Specifically,the organic nutrient sources include glucose and sterile pretreatedmolasses (i.e., molasses converted to reduced sugars), and suitableamounts of carbon sources may be used without limitation.

Nitrogen sources that may be used in the present invention includeinorganic nitrogen sources such as ammonia, ammonium sulfate, ammoniumchloride, ammonium acetate, ammonium phosphate, ammonium carbonate, andammonium nitrate; amino acids such as glutamic acid, methionine andglutamine; and organic nitrogen sources such as peptone, NZ-amine, meatextract, yeast extract, malt extract, corn steep liquor, caseinhydrolysate, fish meal or its digested product, defatted soybean cake orits digested product, etc. These nitrogen sources may be used alone orin combination. The medium may contain potassium phosphate monobasic,potassium phosphate dibasic and corresponding sodium-containing salts,as phosphorus sources.

Inorganic compounds that may be used in the present invention includesodium chloride, calcium chloride, iron chloride, magnesium sulfate,iron sulfate, manganese sulfate and calcium carbonate. In addition, themedium may contain amino acids, vitamins and suitable precursors. Thesemedia or precursors may be added to the medium in a batch or continuousmanner.

Compounds such as ammonium hydroxide, potassium hydroxide, ammonia,phosphoric acid and sulfuric acid may be added to the medium in asuitable manner during culture to adjust the pH of the culture medium.

In addition, during culture, a defoaming agent such as fatty acidpolyglycol ester may be used to suppress the formation of bubbles.Further, in order to maintain the culture medium in an aerobic state,oxygen or oxygen-containing gas may be injected into the culture medium.In addition, in order to maintain the culture medium in an anaerobic ornon-aerobic state, no gas is injected, or nitrogen, hydrogen or carbondioxide gas may be injected into the culture medium. The culture mediumis typically maintained at a temperature ranging from 27° C. to 37° C.,and specifically from 30° C. to 35° C. Culture of the microorganism canbe continued until the desired level of the useful substance will beobtained. Specifically, the culture period is from 10 to 100 hours.

The method of the present invention may further comprise purifying orrecovering the L-amino acid produced in the culture step. Thepurification or recovery process can be performed by purifying orrecovering the desired L-amino acid from the culture medium using asuitable method selected depending on the method used for culture of themicroorganism, for example, a batch, continuous or fed-batch culturemethod.

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes and are not intended to limit thescope of the present invention.

EXAMPLES Example 1: Construction of L-Threonine and L-TryptophanProducing Strains Having Attenuated Activity of Protein that is Encodedby ysaA, ydaS or ybiX Gene

In this Example, each of the ysaA, ydaS and ybiX gene in theL-tryptophan producing strain KCCM10812P (Korean Patent Registration No.10-0792095) and the L-threonine producing strain KCCM10541 (KoreanPatent Registration No. 10-0576342) was deleted by homologousrecombination.

The L-tryptophan producing parent strain KCCM10812P is a strain derivedfrom an E. coli variant KFCC 10066 having L-phenylalanine productivity.It is a recombinant E. coli strain having L-tryptophan productivity,characterized in that chromosomal tryptophan auxotrophy wasdesensitized, the pheA, trpR, mtr and tnaAB genes were attenuated andthe aroG and trpE genes were modified.

Also, the L-threonine producing parent strain KCCM10541 is a strainderived from E. coli KFCC10718 (Korean Patent Laid-Open Publication No.1992-0008365). It has resistance to L-methionine analogue, a methionineauxotroph phenotype, resistance to L-threonine analogue, a leakyisoleucine auxotroph phenotype, resistance to L-lysine analogue, andresistance to α-aminobutyric acid, and is capable of producingL-threonine.

The ysaA, ydaS and ybiX genes to be deleted have the polynucleotidesequences represented by SEQ ID NOS: 1, 2 and 3, respectively.

For this purpose, the one-step inactivation method (developed byDatsenko K A et al.) that is a mutagenesis technique using lambda redrecombinase was used (Proc Natl Acad Sci USA., (2000) 97: 6640-6645). Asa marker for confirming insertion into the genes, thechloramphenicol-resistant gene of pUCprmfmloxC was used (Korean PatentLaid-Open Publication No. 2009-0075549).

About 1200-bp gene fragments were amplified by polymerase chain reaction(hereinafter referred to as PCR) using pUCprmfmloxC as a template and apair of primers 1 and 2, a pair of primers 7 and 8 and a pair of primers13 and 14, which have a portion of each of the three genes and a portionof the chloramphenicol-resistant gene of pUCprmfmloxC. The PCR wasperformed for 30 cycles, each consisting of denaturation at 94° C. for30 sec, annealing at 55° C. for 30 sec and extension at 72° C. for 1min.

TABLE 1  SEQ Primer ID No. Sequence NO 15′-GTAGGGACGCGCTCTCTGGCACTCTGCTGTTTTAG 7TGCAAAGGAGTGATCAGGTGACACTATAGAACGCG-3′ 25′-GGCATAAACAAAGCGCACTGTTCCGGCGTTGAGAA 8ACGCCGGAAAACGTTTAGTGGATCTGATGGGTACC-3′ 35′-GCTTTGGACAAGTGCCAAAACTTTAACATTTCCTT 9CGTTGGATCAAAGCAGTAGGGACGCGCTCTCTGGC-3′ 45′-ATTGAATTTGGAAGAATTTGTAGGCCGGATAAGGC 10GTTTACGCCGCATCTGGCATAAACAAAGCGCACTG-3′ 5 5′-GAGAGAAAAATCTCCTGAAA-3′ 11 65′-CCTACATGATTTCTGCAATA-3′ 12 7 5′-ATTGCGTTAGGCGTCGCCTAATATTTCTGTGTGTT13 TTTGGAGTTCATTCGAGGTGACACTATAGAACGCG-3′ 85′-ATTCGATGTGCTCATGCTTGATTTTCATGAATCAT 14TTGCCTCTTGATGTTTAGTGGATCTGATGGGTACC-3′ 95′-TTACATTAGGCAATCCCTACCCTTACTGCNTTAGG 15CACAGCCTATTGACAATTGCGTTAGGCGTCGCCTA-3′ 105′-ATTGGCTACCCATGCCTGCCCTTTTTCGGCTGCTA 16GGGCAAACAACACTGATTCGATGTGCTCATGCTTG-3′ 11 5′-TATAGAGCCTTTCTTAATCC-3′ 1712 5′-CGCAGATATTCTTCAGTAAT-3′ 18 135′-CATTTCTGATTCAGATGTGGGGCGCAGGCCCCACT 19TTTTGGAGAAATTGTAGGTGACACTATAGAACGCG-3′ 145′-TGTACAGTTAAGTGTAGCTAATCCAGGGACGAACT 20CGGGCAGTTCAAGCATAGTGGATCTGATGGGTACC-3′ 155′-ACCGTTATCACCCGGGCGAGCCAAGAACCTTCTTG 21CTCACAGCCAATATGCATTTCTGATTCAGATGTGG-3′ 165′-GTCATCGTTAGCCCAACCGGATGCCATATCGACCT 22CCCCATATCAATACTTGTACAGTTAAGTGTAGCTA-3 17 5′-AAAGGTTCAGACGGCGCGGT-3′ 2318 5′-TAAGCGCACGCCAGGAATGG-3′ 24

Also, the DNA fragments obtained by the PCR amplification wereelectrophoresed on 0.8% agarose gel, and then eluted and used astemplates in secondary PCR. Secondary PCR was performed so that the 5′and 3′ terminal regions of the primary DNA fragments had 20 pairs ofcomplementary nucleotide bases. About 1300-bp gene fragments wereamplified by PCR using the eluted primary PCR products as templates anda pair of primers 3 and 4, a pair of primers 9 and 10 and a pair ofprimers 15 and 16, which have include the 5′ and 3′ regions of thegenes. The PCR was performed for 30 cycles, each consisting ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec andextension at 72° C. for 1 min. The DNA fragments obtained by the PCRamplification were electrophoresed on 0.8% agarose gel, and then elutedand used in recombination.

According to the method developed by Datsenko K A et al. (Proc Natl AcadSci USA., (2000) 97:6640 6645), an E. coli strain transformed with apKD46 were made competent, and then transformed with the 1300-bp genefragments obtained by PCR. The resulting strains were selected on LBmedium having resistance to chloramphenicol. PCR was performed using apair of primers 5 and 6, a pair of primers 11 and 12 and a pair ofprimers 17 and 18, and the amplification products had sizes of 1450,1530 and 1640 bp, respectively, and were confirmed that the genes weredeleted.

pKD46 was removed from the primary recombinant strains havingchloramphenicol resistance, and then a pJW168 vector was introduced intothe strains, and the chloramphenicol marker gene was removed from thebacterial cells (Gene, (2000) 247, 255-264). The resulting bacterialcells were about 400-bp, 500-bp and 600-bp amplification productsobtained by a pair of primers 5 and 6, a pair of primers 11 and 12 and apair of primers 17 and 18, and were confirmed that the desired genedeletion was achieved.

According to the above-described method, the L-threonine producingstrains KCCM10541 ΔysaA, KCCM10541 ΔydaS and KCCM10541 ΔybiX wereconstructed. Also, the L-tryptophan producing strains KCCM10812P ΔysaA,KCCM10812P ΔydaS and KCCM10812P ΔybiX were constructed.

Example 2: Construction of Recombinant L-Threonine and L-TryptophanProducing Strains Having Deletion of Two or More of ysaA, ydaS and ybiXGenes

According to the method described in Example, recombinant strains havinga deletion of two or more of the genes were constructed.

A pKD46 vector for using lambda red recombinase was introduced into thestrains having a deletion of any one of the genes, and then the strainswere made competent. Also, gene fragments amplified by PCR to include aportion of the three genes and the chloramphenicol-resistant gene ofpUCprmfmloxC were transformed into different strains having a deletionof one of the genes. The resulting strains were screened on LB mediumhaving chloramphenicol resistance, and deletion of a combination of thegenes was confirmed by the use of the primer pairs described in Example1.

According to the above-described method, the L-threonine producingstrains KCCM10541 ΔysaA ΔydaS, KCCM10541 ΔydaS ΔybiX, KCCM10541 ΔybiXΔysaA and KCCM10541 ΔysaA ΔydaS ΔybiX were constructed. Also, theL-tryptophan producing strains KCCM10812P ΔysaA ΔydaS, KCCM10812P ΔydaSΔybiX, KCCM10812P ΔybiX ΔysaA and KCCM10812P ΔysaA ΔydaS ΔybiX wereconstructed.

Among the recombinant strains obtained as described above, strainderived from KCCM10541 through deletion of ysaA, ydaS, and ybiX, andstrain derived from KCCM10812P through deleted of ysaA, ydaS, and ybiXwere named “E. coli CA03-4257P” and “E. coli CA04-2002”, respectively,and deposited at the Korean Culture Center of Microorganisms (361-221,Hongje 1-dong, Seodaemun-gu, Seoul, Korea), an international depositoryauthority, on Dec. 29, 2011 under the accession numbers KCCM11243P andKCCM11245P, respectively.

Example 3: Measurement of Levels of ATP in Constructed L-ThreonineProducing Strains and L-Tryptophan Producing Strains

In this Example, the levels of ATP in the strains constructed inExamples 1 and 2 were quantitatively measured.

For this purpose, the method developed by Kiyotaka Y. Hara et al., whichuses luciferase, was used (“An Efficient Method for Quantitativedetermination of Cellular ATP Synthetic Activity”, J Biom Scre, (2006)V11: No. 3: PP 310-17).

Specifically, the strains having different genetic characters werecultured overnight in LB liquid medium containing glucose. Thesupernatant was removed by centrifugation, the bacterial cells werewashed with 100 mM Tris-Cl (pH 7.5), and then treated with PB buffer(permeable buffer: 40% [v/v] glucose, 0.8% [v/v] Triton X-100) for 30minutes to release intracellular ATP. Next, the supernatant was removedby centrifugation, and luciferin as a substrate for luciferase was addedto the cells. The cells were allowed to stand for 10 minutes, and thenluciferase activity in the cells was measured with a luminometer toquantitatively determine the ATP level. The results of the measurementare shown in FIGS. 1 and 2. All the results were recorded as the averageof three repeated experiments.

As can be seen in FIGS. 1 and 2, the levels of ATP in the strainsconstructed from the L-threonine producing strain and the L-tryptophanproducing strain in Examples 1 and 2 all increased. In addition, the ATPlevel was higher in the strains having a deletion of a combination ofthe genes than in the strains having a deletion of one of the gene.

Example 4: Examination of Titer of L-Threonine Producing Strain, whichhas Attenuated Activity of Alone or a Combination of Enzymes that areEncoded by ysaA, ydaS and ybiX Gene, in Glucose-Containing Medium

According to the methods described in Examples 1 and 2, alone or acombination of the ysaA, ydaS and ybiX genes was deleted from theL-threonine producing strain KCCM10541 (Korean Patent Registration No.10-0576342) to increase the intracellular ATP level. The titers of theresulting strains were evaluated using glucose as a carbon source.

Specifically, the strains having different genetic characters werecultured overnight on LB solid medium in an incubator at 33° C. andinoculated by a platinum loop into 25 mL of glucose-containing mediumhaving the composition shown in Table 2 below. Then, the strains wereincubated in an incubator at 33° C. and at 200 rpm for 50 hours. Theresults are shown in Table 3 below. All the results were recorded as theaverage of three flask results.

TABLE 2 Composition Concentration (per liter) Glucose 70 g KH₂PO₄ 2 g(NH₄)₂SO₄ 25 g MgSO₄•H₂O 1 g FeSO₄•H₂O 5 mg MnSO₄•H₂O 5 mg Yeast extract2 g Calcium carbonate 30 g pH 6.8

TABLE 3 Glucose consumption L-threonine Strain OD (g/L)* (g/L)**KCCM10541 23.7 30.3 31.8 KCCM10541 ΔysaA 24.6 33.4 32.2 KCCM10541 ΔydaS23.5 34.7 33.0 KCCM10541 ΔybiX 22.7 33.9 32.7 KCCM10541 ΔysaA ΔydaS 24.935.1 32.9 KCCM10541 ΔydsS ΔybiX 24.5 36 33.1 KCCM10541 ΔybiX ΔysaA 25.032.1 33.0 KCCM10541 ΔysaA ΔydaS ΔybiX 26.1 36.9 33.9 *measured at 30hours **measured at 50 hours

As can be seen in Table 3 above, we have demonstrated that the glucoseutilization of the recombinant L-threonine producing E. coli strainsconstructed according to the present invention increased by up to about22% compared to that of the parent strain, and the production ofthreonine of the recombinant strains increased by up to about 7%compared to that in the parent strain. In view of the ATP level shown inFIG. 1, these results indicate that the glucose consumption rate oramino acid productivity of the recombinant strains was increased by theincreased ATP level.

Example 5: Examination of Titer of L-Threonine Producing Strain, whichhas Attenuated Activity of Alone or a Combination of Enzymes that areEncoded by ysaA, ydaS and ybiX Gene, in Sucrose-Containing Medium

According to the methods described in Examples 1 and 2, alone or acombination of the ysaA, ydaS and ybiX genes was deleted from theL-threonine producing strain KCCM10541 (Korean Patent Registration No.10-0576342) to increase the intracellular ATP level. The titers of theresulting strains were evaluated using sucrose as a carbon source.

Specifically, the strains having different genetic characters werecultured overnight on LB solid medium in an incubator at 33° C. andinoculated by a platinum loop into 25 mL of sucrose-containing mediumhaving the composition shown in Table 4 below. Then, the strains wereincubated in an incubator at 33° C. and at 200 rpm for 48 hours. Theresults are shown in Table 5 below. All the results were recorded as theaverage of three flask results.

TABLE 4 Composition Concentration (per liter) Sucrose 70 g KH₂PO₄ 2 g(NH₄)₂SO₄ 25 g MgSO₄•H₂O 1 g FeSO₄•H₂O 5 mg MnSO₄•H₂O 5 mg Yeast extract2 g Calcium carbonate 30 g pH 6.8

TABLE 5 sucrose consumption L-threonine Strain OD (g/L)* (g/L)**KCCM10541 26.2 40.0 37.0 KCCM10541 ΔysaA 27.1 40.9 37.5 KCCM10541 ΔdaS26.7 41.7 37.8 KCCM10541 ΔybiX 25 41.1 38.1 KCCM10541 ΔysaA ΔydaS 27.542.1 38.0 KCCM10541 ΔdsS ΔybiX 27.2 43.0 38.1 KCCM10541 ΔybiX ΔysaA 28.342.8 38.7 KCCM10541 ΔysaA ΔydaS ΔybiX 27.9 43.9 38.9 *measured at 24hours **measured at 48 hours

As can be seen in Table 5 above, we have demonstrated that the sucroseutilization of the recombinant L-threonine producing E. coli strainsconstructed according to the present invention increased by up to about10% compared to that of the parent strain, and the production ofthreonine of the recombinant strains increased by up to about 5%compared to that in the parent strain. In view of the ATP level shown inFIG. 1, these results indicate that the activity and sucrose consumptionrate or amino acid productivity of the recombinant strains wereincreased by the increased ATP level.

Example 6: Examination of Titer of L-Tryptophan Producing Strain, whichhas Attenuated Activity of Alone or a Combination of Enzymes that areEncoded by ysaA, ydaS and ybiX Gene, in Glucose-Containing Medium

According to the methods described in Examples 1 and 2, alone or acombination of the ysaA, ydaS and ybiX genes was deleted from theL-tryptophan producing strain KCCM10812P (Korean Patent Registration No.10-0792095) to increase the intracellular ATP level. The titers of theresulting strains were evaluated using glucose as a carbon source.

In order to examine the titer, the strains were inoculated by a platinumloop on LB solid medium and then cultured overnight in an incubator.And, it was inoculated by a platinum loop into 25 mL of flask titermedium having the composition shown in Table 6 below. Then, the strainswere incubated in an incubator at 37° C. and at 200 rpm for 48 hours.The results are shown in Table 7 below. All the results were recorded asthe average of three flask results.

TABLE 6 Composition Concentration (per liter) Glucose 60 g  K₂HPO₄ 1 g(NH₄)₂SO₄ 10 g  NaCl 1 g MgSO₄•H₂O 1 g Sodium citrate 5 g Yeast extract2 g Calcium carbonate 40 g  Sodium citrate 5 g Phenylalanine 0.15 g  Tyrosine 0.1 g   PH 6.8

TABLE 7 Glucose consumption L-tryptophan Strain OD (g/L)* (g/L)**KCCM10812P 18.2 47.2 5.7 KCCM10812P ΔysaA 18.3 48.3 6.9 KCCM10812P ΔydaS18 49.1 6.6 KCCM10812P ΔybiX 17.7 50 6.0 KCCM10812P ΔysaAΔydaS 17.9 48.47.5 KCCM10812P ΔydaSΔybiX 18.7 49.3 7.6 KCCM10812P ΔybiXΔysaA 19.9 497.3 KCCM10812P ΔysaAΔydaSΔybiX 18.9 51.9 7.9 *measured at 33 hours**measured at 48 hours

As can be seen in Table 7 above, we have demonstrated that the glucoseconsumption of the recombinant L-threonine producing E. coli strainsconstructed according to the present invention increased by up to about10% compared to that of the parent strain, and the production oftryptophan of the recombinant strains increased by up to about 38%compared to that in the parent strain. In view of the ATP level shown inFIG. 2, these results indicate that the activity and glucose consumptionrate or amino acid productivity of the recombinant strains wereincreased by the increased ATP level.

While the present invention has been described with reference to theparticular illustrative embodiments, those skilled in the art to whichthe present invention pertains can understand that the present inventionmay be embodied in other specific forms without departing from thetechnical spirit or essential characteristics of the present invention.Therefore, the embodiments described above are considered to beillustrative in all respects and not restrictive. Furthermore, the scopeof the present invention is defined by the appended claims rather thanthe detailed description, and it should be understood that allmodifications or variations derived from the meanings and scope of thepresent invention and equivalents thereof are included in the scope ofthe appended claims.

Accession Number

Depository authority: Korean Culture Center of Microorganisms(International)

Accession Number: KCCM11243P

Deposition date: Dec. 29, 2011

Depository authority: Korean Culture Center of Microorganisms(International)

Accession Number: KCCM11245P

Deposition date: Dec. 29, 2011

The invention claimed is:
 1. An L-threonine or L-tryptophan producingrecombinant Escherichia coli strain, wherein the strain is modified toattenuate activity of at least two selected from the group consistingof: a protein YsaA having the amino acid sequence of SEQ ID NO: 2, aprotein YdaS having the amino acid sequence of SEQ ID NO: 4, and aprotein YbiX having the amino acid sequence of SEQ ID NO: 6, wherein theactivity is attenuated by deletion of at least a gene encoding theprotein YsaA, the protein YdaS, and/or the protein YbiX.
 2. TheL-threonine or L-tryptophan producing recombinant Escherichia colistrain according to claim 1, wherein the protein YsaA is encoded by apolynucleotide sequence of SEQ ID NO: 1, the protein YdaS is encoded bya polynucleotide sequence of SEQ ID NO: 3, and the protein YbiX isencoded by a polynucleotide sequence of SEQ ID NO:
 5. 3. The L-threonineor L-tryptophan producing recombinant Escherichia coli strain accordingto claim 1, wherein the recombinant Escherichia coli strain isL-threonine producing Escherichia coli CA03-4257P (KCCM11243P).
 4. TheL-threonine or L-tryptophan producing recombinant Escherichia colistrain according to claim 1, wherein the recombinant Escherichia colistrain is L-tryptophan producing Escherichia coli CA04-2002(KCCM11245P).
 5. A method for producing L-threonine or L-tryptophan, themethod comprising culturing an L-threonine or L-tryptophan producingrecombinant Escherichia coli strain, wherein the strain is modified toattenuate activity of at least two selected from the group consisting ofa protein (YsaA) having the amino acid sequence of SEQ ID NO: 2, aprotein (YdaS) having the amino acid sequence of SEQ ID NO: 4, and aprotein (YbiX) having the amino acid sequence of SEQ ID NO: 6, whereinthe activity is attenuated by deletion of at least a gene encoding theprotein YsaA, protein YdaS, and/or the protein YbiX.
 6. The methodaccording to claim 5, wherein the recombinant Escherichia coli strain isL-threonine producing Escherichia coli CA03-4257P (KCCM11243P).
 7. Themethod according to claim 5, wherein the recombinant Escherichia colistrain is L-tryptophan producing Escherichia coli CA04-2002(KCCM11245P).